CN114854734B - Pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set and kit thereof - Google Patents
Pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set and kit thereof Download PDFInfo
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Abstract
The invention relates to a pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set and a kit. The primer group is designed according to VP6 gene of pigeon rotavirus A type: outer primers (PiRVA-F3 and PiRVA-B3), inner primers (PiRVA-FIP and PiRVA-BIP) and loop primers (PiRVA-LB). The specific sequences are shown in SEQ ID NO.1-5 respectively. The invention establishes a pigeon rotavirus A-type loop-mediated isothermal amplification detection method according to the designed primer group, and the detection method does not cross react with the pathogen of the pigeon common infectious diseases. The detection method has the advantages of strong specificity, good accuracy and high sensitivity, does not need expensive instruments and equipment, is convenient for clinical field use of the basic level, and is convenient and quick.
Description
Technical Field
The invention belongs to the field of poultry pathology detection, and particularly relates to a pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set and a kit.
Background
The pigeons are domesticated into ornamental pigeons, racing pigeons and meat pigeons, have a strong immune system, and the pigeons are mostly open or semi-open, so that the pigeons have good air circulation and fresh air, and are less ill compared with other livestock and poultry. However, with the rapid development of large-scale and intensive pigeon raising industry, the raising total amount and raising density are increased, the pigeon raising mode is changed, and due to the low level of raising management and poor awareness of epidemic disease prevention and control, and the frequent trade circulation of pigeons (including the competition of carrier pigeons), the pigeon diseases are more and more serious and more complex. In recent years, the pigeon viral infectious diseases seriously threaten the pigeon raising industry, and according to domestic and foreign researches, the reported pigeon viral infectious diseases comprise pigeon newcastle disease, pigeon rotavirus infection, pigeon adenovirus infection, pigeon circovirus infection, pigeon herpes virus infection, pigeon pox, H9 subtype low-pathogenicity avian influenza and the like.
Rotavirus (RV) belongs to the genus Rotavirus of the family reoviridae, and the virion is non-enveloped, is in the form of a regular icosahedron, is divided into 3 layers of shells, has a diameter of about 75nm, and is in the form of a wheel under electron microscopy, thus obtaining the name. The rotavirus genome consists of 11 segments of double-stranded RNA, and each segment is 663-3302 bp long. According to the latest classification by the international committee on classification of viruses (International Committee on Taxonomy of Viruses, ICTV), rotavirus comprises a total of 10 virus species a to J. To date, rotavirus A, D, F and G can infect birds, and rotavirus A, D and G can infect pigeons. The differences in gene sequences between strains of different hosts of the same virus species are often significant. Rotavirus type a (RVA) typically infects animals of a specific population, such viruses are known as homologous strains, but occasionally there is also cross-species transmission. Rotavirus type a hosts are widely present in mammals and birds, and clinical symptoms after infection of birds are mainly manifested by severe diarrhea, dehydration, dysplasia and increased mortality. Infection of pigeons with rotavirus type a is characterized by vomiting, diarrhea, dehydration and cloaca. The ill pigeon suffers from poor spirit, anorexia and digestive dysfunction. Then, the patient is listlessness, sleepiness, inappetence and severe diarrhea, the excrement is watery, severely dehydrated, thin, anemias and finally dies due to failure. The lesions of the section are mainly manifested by necrosis of the liver and congestion, bleeding and oedema of the mucous membrane of the intestine, and in addition, dehydration of the body and inflammation of the cloaca. Rotavirus type a infection has severely affected the healthy development of pigeon raising.
The virus molecular biology detection mainly comprises a conventional PCR method and a real-time fluorescence quantitative PCR method, but is difficult to popularize and apply in the inspection and quarantine departments of the basic level due to higher requirements of experimental equipment. The loop-mediated isothermal amplification (LAMP) technology is a constant-temperature amplification technology for target genes by using 4-6 specific primers and Bst DNA polymerase, and has the characteristics of strong amplification specificity, high sensitivity, rapid and simple operation, simple detection and the like, gets rid of the dependence on expensive instruments such as a PCR instrument, a real-time fluorescent quantitative PCR instrument and the like, and is more convenient for detection in a basic unit. LAMP has a number of unique advantages over other existing nucleic acid amplification techniques: (1) the LAMP detection technology has high amplification specificity, the specific primer can accurately identify the target sequence, and the LAMP detection technology has high selectivity to the target sequence, so that non-specific amplification is reduced. (2) The LAMP technology can realize amplification under isothermal conditions, reduces the requirements on expensive and precise experimental instruments, has high amplification efficiency, only needs a common water bath kettle to regulate the temperature (60-65 ℃), greatly reduces the detection cost, and is particularly suitable for use on a base layer and on site. (3) The LAMP detection technology can generate a large amount of double-stranded DNA mixture and white magnesium pyrophosphate precipitate in positive amplification reaction, the amplified products have various detection methods, the double-stranded DNA products can be detected by agarose gel electrophoresis, the white magnesium pyrophosphate precipitate can be detected by a turbidity meter, or a metal ion complexing agent or a metal ion indicator can be added into a reaction system, and then analysis can be directly carried out according to the change of color or ultraviolet irradiation. At present, no related research report aiming at a pigeon rotavirus A type loop-mediated isothermal amplification reaction method is found, and the establishment of the method can fill the blank of related fields at home and abroad.
Disclosure of Invention
The invention aims to provide a pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set and a kit.
The aim of the invention is realized by the following technical scheme:
a pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set consists of 1 pair of outer primers PiRVA-F3 and PiRVA-B3, 1 pair of inner primers PiRVA-FIP and PiRVA-BIP and 1 loop primer PiRVA-LB, wherein the sequences of the primers are as follows:
PiRVA-F3：5’-AAATCACGCAACAGTCGG-3’，
PiRVA-B3：5’-AGTTATCTTCTCTAGATGCTGAA-3’，
PiRVA-FIP：5’-GACAAATATGGCTCATTTGCATCTG-CTGACACTAAAAATTGAATCCG-3’，
PiRVA-BIP：5’-ACAGGTTTGAGACAGGAATACGC-AATTATTAAGCAACTCAGTCCA-3’，
PiRVA-LB：5’-GATTCCAGTAGGACCAGTATTTCC-3’。
a pigeon rotavirus A-type detection kit containing the primer group.
The LAMP reaction system of the pigeon rotavirus A type detection kit comprises 20 mu L: after dissolution, LAMP OG Reagent 15. Mu.L, LAMP Primer Mix 2. Mu.L, template RNA 1. Mu. L, ddH 2 O2. Mu.L; wherein LAMP Primer Mix concentration: piRVA-FIP and PiRVA-BIP were 8. Mu. M, piRVA-LB 4. Mu. M, piRVA-F3 and PiRVA-B3 were 1. Mu.M respectively; the reaction conditions are as follows: 30min at 63 ℃.
The application of the pigeon rotavirus A type loop-mediated isothermal amplification detection primer group in preparation of pigeon rotavirus A type detection kit.
The application of the pigeon rotavirus A type loop-mediated isothermal amplification detection primer group in preparing a pigeon rotavirus A type rapid diagnostic reagent.
Compared with the prior art, the invention has the advantages that:
according to the gene sequence of the pigeon rotavirus A, and the whole gene comparison analysis, the invention selects the VP6 gene of the pigeon rotavirus A to design a specific LAMP primer, and establishes a loop-mediated isothermal amplification method for detecting the pigeon rotavirus A according to the designed LAMP primer. The method can only perform specific reaction with the pigeon rotavirus A (only the pigeon rotavirus A presents macroscopic green), and does not perform specific reaction with other common infectious diseases (PiAdVB, piCV, piNDV, piHV and H9 AIV) (other pathogens present macroscopic orange), so that the method is high in specificity, high in accuracy and good in applicability.
The sensitivity experiment shows that the detection method established by the invention can detect the virus nucleic acid with the concentration as low as 0.184 ng/. Mu.L, which indicates that the LAMP method has higher sensitivity.
After the LAMP reaction established by the invention is finished, an EP reaction tube is arranged in the normal direction without uncovering, and the reaction liquid is thrown to the bottom of the EP tube, so that the result judgment is directly carried out according to the color change, the pollution possibility is greatly reduced, and the usability of the method is improved.
Drawings
FIG. 1 is a diagram showing the results of a specific test of the LAMP detection method established in the present invention, wherein 1-2 is Pigeon rotavirus A (PiRVA); 3 is pigeon adenovirus type B (PiAdVB); 4 is pigeon circovirus (Picv); 5 is pigeon Paramyxovirus (PiNDV); 6 is pigeon herpesvirus (PiHV); 7 is H9 subtype avian influenza virus (H9 AIV); 8 is ddH 2 O。
FIG. 2 is a graph showing the results of a sensitivity test of the LAMP detection method established in the present invention. Wherein 1 is 1.84X10 1 ng/. Mu.L; 2 is 1.84×10 0 ng/. Mu.L; 3 is 1.84×10 -1 ng/. Mu.L; 4 is 1.84×10 -2 ng/. Mu.L; 5 is 1.84×10 -3 ng/. Mu.L; 6 is 1.84×10 -4 ng/. Mu.L; 7 is 1.84×10 -5 ng/. Mu.L; 8 is ddH 2 O。
Detailed Description
The invention is further described below, the embodiments presented in this description are only exemplary and do not limit the scope of the invention. It will be understood by those skilled in the art that the details and forms of the invention may be modified or substituted without departing from the spirit and scope of the invention.
The invention provides a pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set, which consists of 1 pair of outer primers PiRVA-F3 and PiRVA-B3, 1 pair of inner primers PiRVA-FIP and PiRVA-BIP and 1 loop primer PiRVA-LB, wherein the sequences of the primers are as follows:
PiRVA-F3：5’-AAATCACGCAACAGTCGG-3’，
PiRVA-B3：5’-AGTTATCTTCTCTAGATGCTGAA-3’，
PiRVA-FIP：5’-GACAAATATGGCTCATTTGCATCTG-CTGACACTAAAAATTGAATCCG-3’，
PiRVA-BIP：5’-ACAGGTTTGAGACAGGAATACGC-AATTATTAAGCAACTCAGTCCA-3’，
PiRVA-LB：5’-GATTCCAGTAGGACCAGTATTTCC-3’。
the primer group provided by the invention can be used for preparing a pigeon rotavirus A-type detection kit and/or a rapid diagnostic reagent.
The loop-mediated isothermal amplification detection method of the pigeon rotavirus A detection kit comprises 20 mu L of reaction system, wherein each 20 mu L of reaction system comprises the following steps: after dissolution, LAMP OG Reagent 15. Mu.L, LAMP Primer Mix 2. Mu.L, template RNA 1. Mu. L, ddH 2 O2. Mu.L; wherein LAMP Primer Mix concentration: piRVA-FIP and PiRVA-BIP were 8. Mu. M, piRVA-LB 4. Mu. M, piRVA-F3 and PiRVA-B3 were 1. Mu.M respectively;
the established reaction conditions of the loop-mediated isothermal amplification detection method are as follows: 30min at 63 ℃.
The invention will be described in more detail with reference to the following examples:
example 1
1. Experimental method
1. Test strains
The pathogenic pigeon rotavirus type A (PiRVA), pigeon adenovirus type B (PiAdVB), pigeon circovirus (PiCV), pigeon Paramyxovirus (PiNDV), pigeon herpesvirus (PiHV), and H9 subtype avian influenza virus (H9 AIV) used in the test were all identified and stored by the institute of livestock and veterinary at the national academy of sciences of Fujian and agriculture.
2. Preparation of nucleic acids
Extracting the relevant viral nucleic acid with commercial nucleic acid extraction kit instructions. Wherein PiRVA, piNDV, H AIV extracts viral nucleic acid RNA; piAdVB, piCV, piHV viral nucleic acid DNA is extracted.
Primer design for LAMP
According to the gene sequences of all pigeon rotavirus A types registered in GenBank, and performing total gene comparison analysis, VP6 gene design primers of pigeon rotavirus A types are selected, wherein the primers comprise 1 pair of outer primers (PiRVA-F3 and PiRVA-B3), 1 pair of inner primers (PiRVA-FIP and PiRVA-BIP) and 1 pair of ring primers (PiRVA-LB), and specific sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.
Establishment of LAMP reaction
LAMP test reaction solution was prepared in a 0.2mL EP reaction tube according to the specification of a freeze-dried LAMP orange green color-changing amplification kit (the reagent contains bst4.0 DNA polymerase, either DNA or RNA) and the reaction system was 20 μl, and each 20 μl of the reaction system contains: after dissolution, LAMP OG Reagent 15. Mu.L, LAMP Primer Mix 2. Mu.L, template RNA or DNA 1. Mu. L, ddH 2 O2. Mu.L. After all reagents are added, the bottom of the EP tube is flicked, and then the EP tube cover containing the OG dye is covered (the OG dye on the tube cover cannot be mixed and inverted vigorously after the tube cover is covered, so that the problem that the OG dye on the tube cover is dissolved and the LAMP reaction is stopped once the OG dye is mixed into the reaction solution is solved). After the reaction system is prepared, the mixture is placed at 60-65 ℃ for reaction for 20-45 min. After the reaction was completed, the EP tube was inverted and allowed to stand for 30 seconds. The EP tube was then set up and the reaction solution was gently flung to the bottom of the EP tube, at which time the positive amplified sample would become visually green, while the negative non-amplified tube would be orange.
4.1 optimization of LAMP method
The invention optimizes the concentration of the LAMP Primer Mix, and the optimized optimal LAMP Primer Mix concentration is as follows: piRVA-FIP/PiRVA-BIP of 8. Mu. M, piRVA-LB of 4. Mu. M, piRVA-F3/PiRVA-B3 of 1. Mu.M, respectively.
The invention optimizes the reaction temperature and time by detecting the reaction performance of the primer group at different temperatures (60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃) and different times (20 min, 25min, 30min, 35min, 40min and 45 min) through experiments. As a result, it was found that the optimal reaction condition after optimization was 63℃for 30 minutes.
Specificity test of LAMP detection method
Nucleic acid DNA or RNA of pigeon rotavirus A (PiRVA), pigeon adenovirus B (PiAdVB), pigeon circovirus (PiCV), pigeon Paramyxovirus (PiNDV), pigeon herpesvirus (PiHV) and H9 subtype avian influenza virus (H9 AIV) are taken and detected by established LAMP methods (reaction using optimized LAMP conditions) respectively. The results indicate that only pigeon rotavirus type a (PiRVA) appears macroscopic green and the other pathogens (PiAdVB, piCV, piNDV, piHV, H AIV) all appear macroscopic orange (see figure 1). The LAMP detection method established by the invention can be judged through obvious color distinction under the natural light condition, which shows that the established LAMP method has good specificity and applicability.
Sensitivity test of LAMP detection method
The RNA concentration of extracted pigeon rotavirus A (PiRVA) is measured to be 18.4 ng/. Mu.L by using a Nanodrop 2000 spectrophotometer, the RNA solution is diluted by 10 times, 7 dilution gradients are arranged in the stock solution, the RNA with the 7 dilution gradients is used as templates respectively, and the detection is carried out by using an established LAMP method (the reaction is carried out by adopting the optimized LAMP condition). The results show (as shown in FIG. 2), that the established LAMP method can detect sample 10 2 Dilutions, i.e., 0.184 ng/. Mu.L RNA. The sensitivity of the LAMP method was shown to be high.
7. Clinical testing application
After grinding the collected 33 pigeon tissue disease materials, extracting virus nucleic acid according to a commercial kit, and detecting by using an established LAMP method. The result shows that 6 samples are visible green (namely, pigeon rotavirus A infection is positive) after detection, and the positive rate is 18.18% (6/33).
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Sequence listing
<110> institute of livestock and veterinary at the national academy of agricultural sciences of Fujian province
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Claims (1)
1. A pigeon rotavirus A-type detection kit containing a pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set is characterized in that: the reaction system of the kit is 20 mu L, and the 20 mu L reaction system comprises: after dissolution, LAMP OG Reagent 15. Mu.L, LAMP Primer Mix 2. Mu.L, template RNA or DNA 1. Mu. L, ddH 2 O 2μL；
Wherein, the concentration of each Primer in LAMP Primer Mix is as follows: piRVA-FIP and PiRVA-BIP were 8. Mu. M, piRVA-LB 4. Mu. M, piRVA-F3 and PiRVA-B3 were 1. Mu.M respectively;
the reaction conditions are as follows: 30min at 63 ℃;
wherein, the pigeon rotavirus A-type loop-mediated isothermal amplification detection primer group consists of 1 pair of outer primers PiRVA-F3 and PiRVA-B3, 1 pair of inner primers PiRVA-FIP and PiRVA-BIP, and 1 loop primer PiRVA-LB, and the sequences of the primers are as follows:
PiRVA-F3：5’-AAATCACGCAACAGTCGG-3’，
PiRVA-B3：5’-AGTTATCTTCTCTAGATGCTGAA-3’，
PiRVA-FIP：5’-
GACAAATATGGCTCATTTGCATCTG-CTGACACTAAAAATTGAATCCG-3’，
PiRVA-BIP：5’-
ACAGGTTTGAGACAGGAATACGC-AATTATTAAGCAACTCAGTCCA-3’，
PiRVA-LB：5’-GATTCCAGTAGGACCAGTATTTCC-3’。
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